Biologically active macromolecules are complex entities which have intricate three dimensional structures. The exact spatial conformation of these macromolecules is directly related to their biological function. For example, proteins are composed of amino acids in well defined sequences. Amino acids may have both polar and nonpolar side groups. The polar side groups may in turn be either charged or uncharged in aqueous buffers near neutral physiological pH.
The three dimensional structure of a particular polypeptide chain depends on the multiple spatially specific interactions of its unique sequence, or primary structure. The chain folds into a three dimensional structure in accordance with the multitude of specific interactions among the constituent amino acid units. For example, there are 1026 possible primary structures for a 20 amino acid polypeptide. The secondary, tertiary, and quaternary structural possibilities increase the complexity.
The human immune system is impressive in its ability to generate and screen approximately 1012 antibody molecules in order to identify one that specifically recognizes and binds a foreign pathogen (A. Nisonoff, J. E. Hopper, S. B. Spring, “The Antibody Molecule”, Academic Press, New York, 1975). The large number of trials is necessitated by the equally large number of sequences available through the twenty or so essential amino acids. For each invading foreign pathogen, there is perhaps only one (or at best a few) optimal backbone sequence that offers the potential for specific ligand-receptor, “lock-and-key” docking or “induced fit” docking. FIG. 1a illustrates lock-and-key docking wherein the active site of an enzyme is complementary in shape and electronic force field to that of the substrate. FIG. 1b illustrates induced fit docking wherein the active site of an enzyme has a shape complementary to that of the substrate only after the substrate is bound. Upon binding the active site of the enzyme is slightly distorted to accommodate the fit.
Combinatorial chemistry seeks to mimic the natural function of the immune system. It too involves examining large compound libraries to find the unique compound for binding to a target molecule (S. P. A. Fodor, et al., Science, 251, 767, 1991). In combinatorial chemistry compound libraries are designed and synthesized. The libraries comprise a large number of macromolecules produced via the combination of a variety of monomer precursors. The libraries are screened for new drugs, for example, antagonists of a specific receptor target. The ability to generate large, random compound libraries is important in combinatorial chemistry because the greater the diversity the more likely the library will include all possible monomer combinations that could react with or bind the target.
A great deal of effort is being spent on the application of these combinatorial libraries, as well as libraries of small organic molecules, for the discovery of new drugs (B. A. Bunin, M. J. Plunkett, J. A. Ellman, Proc. Natl., Acad. Sci., U.S.A., 91, 4708, 1994). Libraries may ranging in size and can include, for example, from 100,000 to 500,000 compounds per library.
Despite its power, combinatorial chemistry suffers from several intrinsic weaknesses. First, it is a statistical approach. For example, a shot-gun or hit-or-miss approach is used for material discovery. Hundreds of thousands of compounds must be synthesized and screened to find a compound that may react with or bind the target. While combinatorial chemistry involves single-chain synthesis, the chain building process is inefficient. Even if the synthetic yield of attaching one repeat unit to a growing chain is 99%, the composite yield of growing a chain consisting of ten repeat units is only 90.4%, of twenty repeat units is only 82.6% and of thirty repeat units is only 75.5%.
The inefficient chain building process restricts single chains to be individually synthesized with limited chain lengths. Therefore, the three dimensional topography of the resulting coils can only be empirically sampled by studying the binding behavior with probe molecules. Binding requiring the coordinated interaction with more than ten repeat units is very difficult to establish because of the chain building inefficiency described above.
Additionally, unencumbered folding is not achieved because the chains are attached at one end to a substrate during synthesis. The lack of precise knowledge of coil conformation dependence on chain sequence precludes accurate prediction of folding pathways and biologically active site topography, where folding occurs in the test macromolecules.
Finally, the three dimensional structure of the folded proteins is held together by primarily nonbonded interactions. Hence, such a structure is delicate and susceptible to thermal and non-aqueous solvent induced denaturing. Peptide linkages also are susceptible to biological degradation in vivo through the intervention of digestive enzymes, for example, protease and chymotrypsin. Therefore, combinatorial chemistry is disadvantageous in terms of the time, money and effort required to produce the libraries and test them.
Whereas combinatorial chemistry and the immune system rely on a “shotgun” approach to find complements to targets, molecular imprinted polymers (MIPs) rely on a semi-directed method of forming recognition sites. See (a) Mosbach, K., Trends Biochem. Sci., 19:9-14 (1994), (b) Wulff, G., Angew. Chem. Int. Ed. Engl., 34:1812-1832 (1995) (c) Mosbach, K. and Ramström, O; Biotechnology, 14:163-170 (1996) and (d) Takeuchi, T. and Matsui, J. Acta Polymer, 47:471-480 (1996). MIPs are fabricated by polymerizing monomers (e.g., acrylic acid) and cross-linking molecules (e.g., ethylene glycol diacrylate) in the presence of an “imprinting molecule” to produce a large, rigid, and insoluble polymer structure. A nonsolvent (e.g., chloroform) “porogen” is employed during polymerization to produce large pores in the bulk material. The porogen is required to allow target diffusion into the insoluble MIP's inner regions, where the large percentage of the imprints are located. MIP structures must be rigid to achieve target recognition. This rigidity is achieved by employing crosslinking molecules that have a high number of crosslinking moieties per molecular weight. Both bulk and suspension polymerization schemes have been employed. Bulk polymerization produces a rigid plastic product the size of the reaction container, while a polymeric sphere on the order of a micrometer in size (a microsphere) is formed through suspension polymerization.
The resulting MIP is a porous macroscopic solid, which is not soluble and due to its size it settles out of solution. The porous nature of the MIP structure produces diffusional resistance, reducing separation efficiency. Each MIP has many imprint sites (1023) with widely varying degrees of binding affinity with only a small fraction of sites accessible to targets. The use of the porogen in the synthesis procedure results in large voids throughout the structure. The voids are intended to allow for diffusion of target molecules into and out of the imprinted sites. However, many sites are still inaccessible.
MIPs have been used in chromatography (O'Shannessy, D. J, et al., Journal of Molecular Recognition, 2:1-5 (1989); Fischer, L., et al., PCT WO 93/09075; and Yoshikawa et al., Polymer Journal, 29:205-210 (1997)), as artificial antibodies (Arshady, R. and Mosbach, K., Makromol. Chem., 182:687-692 (1981); Ekberg, B., Mosbach, K. Tibtech, 7:92-96 (1989); Mosbach, K., U.S. Pat. No. 5,110,833; Vlatakis, G., et al., Nature, 361:645-647 (1993); Glad, M., et al., PCT WO 93/05068; Shimada, T., et al., Bull. Chem. Soc. Jpn. 67:227-235 (1994); Mosbach, K., PCT WO 94/11403; Kriz, et al., Anal. Chem. 67:2142-2144 (1995); Yan, M. Y., et al., U.S. Pat. No. 5,587,273; Haupt, K., et al., 213th ACS National Meeting Abstracts, Section I&EC, 1997, No. 32; and Mosbach, K., et al., PCT WO 97/22366), and as artificial enzymes (Wulff, G., et al., U.S. Pat. No. 4,127,730; Kempe, M. and Mosbach, K. Analytical Letters, 24:1137-1145 (1991); and Tanaka, T., et al., PCT WO 94/26381).
MIP's used for chromatography are often used for separating chiral isomers. An important property of biologically active compounds is their stereochemistry. For example, most of the essential amino acids are chiral, having L- and D-isomers. However, only the L-isomers are found in proteins. In general, biological activity implies optically specific interactions. A docking site (active, catalytic, or regulatory site) is commonly defined by multiple interaction points (force fields) that are inherently asymmetric (optically active). Pharmaceuticals, agrochemicals, flavors, and fragrances contain many naturally occurring or synthetic stereoisomers. There has been much attention given to the development of methods for separating isomers, among them MIP's and enzymatic kinetic resolution. There is an important need in the field for effective methods of resolving chiral isomers. There is a need for efficient methods for producing molecules which are capable of interacting with specific targets. There further is a need for the development of molecules capable of binding specifically with target compounds which can be used to separate the target compounds, or which can be used as synthetic antibody or enzyme mimics or any of a variety of applications where specific interaction with a target molecule occurs. There also is a need for improved methods for isolating and purifying optical isomers, and for improved affinity materials that preferentially interact with a desired isomer.